Increased sulfatation of orbital glycosaminoglycans in Graves' ophthalmopathy.

Accumulation of interstitial glycosaminoglycans (GAG) in orbital tissue of patients with Graves' ophthalmopathy (GO) leads to edema, increased orbital pressure, and proptosis. In this study, a new, highly sensitive, high performance liquid chromatography method was developed to determine the altered concentration and biochemical composition of different GAG polymers in orbital connective tissue of 27 GO patients and 18 controls. GAG were isolated by tissue homogenization and digestion, followed by sequential enzymatic GAG hydrolysis and high performance liquid chromatographic analysis of the resulting alpha,beta-unsaturated disaccharides. High recovery rates of 78 +/- 6% (mean +/- SE) and a detection limit of 4.0 microg/L (0.01 micromol/L) were obtained. Total tissue GAG amounted to 254 +/- 16 microg/g wet tissue wt in patients and 150 +/- 13 microg/g (P < 0.0001) in controls. Regarding the GAG polymers, marked differences were detected between patients and controls (chondroitin sulfate, 127 +/- 13 vs. 47 +/- 5 microg/g; hyaluronic acid, 56 +/- 5 vs. 34 +/- 4 microg/g; both P < 0.0001; dermatan sulfate, 77 +/- 6 vs. 69 +/- 6 microg/g; P < 0.05). In patients, chondroitin sulfate was the major GAG component (48 +/- 6 vs. 31 +/- 5% of total GAG in controls), whereas dermatan sulfate was dominant in controls (46 +/- 8% vs. 30 +/- 5%). The sulfated disaccharide digestion products were markedly increased (P < 0.0001) in patients, and the ratio of sulfated vs. total disaccharide content was 85 +/- 6% vs. 65 +/- 5% (P < 0.05) in patients and controls, respectively. As accumulation of negatively charged sulfate residues in GAG disaccharides results in enhanced water-binding capacity, beside inflammation and increased volume of the orbital adipose tissue, the altered structure and nature of sulfated GAG units in the orbit may be responsible for the pathogenic changes in Graves' ophthalmopathy.

HPLC glycosaminoglycan analysis in patients with Graves' disease.

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAGs) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with Graves' eye disease. 2. A highly specific method to determine the concentration and biochemical composition of different GAGs was developed in order to obtain a sensitive test system for the activity of the disease. By means of this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chloride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyaluronate lyase, with HPLC analysis of the resulting alpha, beta-unsaturated disaccharides by anion-exchange chromatography. 3. Concentrations of GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluronic acid (HA) were determined in patients and controls, with high recovery rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 micrograms/l (0.01 mumol/l)], revealing marked differences in urinary concentrations of total GAG and HA, as well as an elevation of CA in patients compared with controls. 4. Method sensitivity was 0.86 for patients with active Graves' eye disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; CA, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; during therapy: GAG, 54.22 +/- 10.94; CA, 20.52 +/- 4.58; DS, 17.65 +/- 3.46; HA, 16.05 +/- 3.69 mg/24 h), whereas GAG excretion increased markedly 2-3 months after stopping prednisone therapy in patients with still active eye disease (GAG, 109.9 +/- 10.51; CA, 63.8 +/- 7.34; DS, 24.1 +/- 5.07; HA, 22.0 +/- 6.28 mg/24 h). 5. This sensitive method determines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.